Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding Sequence Listing and the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
Catalytic antibodies have unique potential for the treatment of cocaine addiction and overdose. Cocaine reinforces self-administration by inhibiting a dopamine re-uptake transporter (1) in the mesolimbocortical "reward pathway". No antagonist to cocaine is known (2), perhaps reflecting the difficulties inherent in blocking a blocker. As an alternative to receptor-based therapeutics, a circulating agent could interrupt the delivery of cocaine to its binding site in the brain (3). An agent such as an antibody that merely bound the drug could be depleted stoichiometrically by complex formation but an enzyme that bound drug, transformed it and released product would be available for additional binding. Catalytic antibodies, a novel class of artificial enzyme, are inducible for a wide array of reactions and their substrate specificity is programmable to small molecules such as cocaine (4).
Cocaine detoxification is particularly well suited for a catalytic antibody approach. First, hydrolysis of the benzoyl ester of cocaine yields the biologically inactive products (5) ecgonine methyl ester and benzoic acid (FIG. 1). The plasma enzyme butyrylcholinesterase deactivates cocaine in humans (6) by means of this reaction. Second, acyl hydrolysis is the best studied of all antibody-catalyzed transformations (7,8). Esterase activity approaching that of natural enzymes has been reported (7) for catalytic antibodies and the large hydrophobic surface of the benzoyl ester is particularly well suited to elicit antibodies with strong binding and catalysis.
It has previously described (9) the first catalytic antibodies to degrade cocaine, Mab 3B9 and Mab 6A12. The antibodies were elicited by an immunogenic conjugate (TSA 1) of a phosphonate monoester transition-state analog. The rate acceleration of these first artificial cocaine esterases (10.sup.2 -10.sup.3) corresponded in magnitude to their relative stabilization of the ground-state to the transition-state (.about.K.sub.m /K.sub.i). Catalytic antibodies with more potent catalytic mechanisms and with higher turnover rates are possible and, it has been estimated, necessary for clinical applications. Increased activity can be pursued either through repeated hybridoma generation or through mutagenesis of catalytic antibodies in hand. However, sequencing of the variable domains of Mab's 3B9 and 6A12 revealed 93% homology at the complementarity determining regions (see below). Such a lack of diversity has been noted previously for catalytic antibodies (10) and limits the opportunities for improving activity since a particular class of homologous catalytic antibodies may fail to optimize to the desired activity. A potential solution to this problem, that would not compromise the core structure of the analog, would be to vary the surfaces of the analog rendered inaccessible by attachment to carrier protein and thereby present distinct epitopes for immunorecognition.
The syntheses of three analogs of cocaine hydrolysis with identical phosphonate replacements but differing constructions for the immunoconjugates is now reported. The kinetics and the structural diversity of the catalytic antibodies elicited by these analogs has been characterized. The preferred catalytic antibodies for mutagenesis studies have been identified.